Applications of Fluorescence Lifetime Imaging Microscopy

Life Sciences are the main field of application for our proprietary Fluorescence Lifetime Imaging Microscopy (FLIM) devices. Our technology is frequently used in the following domains:

• Molecular Imaging
• Metabolic Imaging
• FRET Imaging
• Simultaneous NAD(P)H and pO2 Imaging

Also, the evaluation of fast physiological effects as well as Stimulated-Emission Depletion (STED) microscopy are possible using this method. The fluorescence lifetime of most fluorophores is depending on their environment and their interactions with it. The measurement of molecular-environment parameters, such as pH, concentration of oxygen, physiologically important ions or proteins, can be crucial for the evaluation of fluorescence lifetime and intensity of the observed fluorophore. Whether observation of the fluorescence lifetimes of a sample with a pH-sensitive fluorescent probe, protein-interaction experiments by Förster Resonance Energy Transfer (FRET) or mesaurements of the Ca2+ concentration in cells: Excellent image and time resolution are key. With our FLIM upgrades for your existing one photon and multiphoton microscope as well as our multiple ready-made FLIM solutions, researchers and scientists are able to perform a number of highly precise analyses on live cells on a molecular level.

FLIM-Based In Vivo Imaging and More Clinical Applications

The methods of Fluorescence Lifetime Imaging Microscopy (FLIM) are particularly suited for in-vivo diagnostics as they are noninvasive and non-destructive. Thus, they are widely used for clinical research and medical examination on live subjects. For instance, the technique can be used in the following fields:

• In-vivo diagnostics of skin: Multiphoton-lifetime tomography of human skin uses two-photons excitation in combination with non-descanned detection.As a result, the multiple-photon FLIM delivers optically sectioned images oftissue layers as deep as 100 µm. In-vivo two-photon imaging of human skin cells is possible without impairing the viability of the tissue. From the sub-cellular resolution, three-dimensional structures can be reconstructed.
• Ophthalmic examinations: Ophthalmic FLIM uses a combination of fast beam scanning and excitation by a picosecond diode laser. This method is so sensitive that it is able to record lifetime images of the fundus (background) of the human eye. By this, the early discovery of eye diseases is possible, as these are often accompanied by metabolic changes of the fundus. In turn, these cause changes in the fluorescence decay parameters of endogenous fluorophores.
• Personalized chemotherapy: To target the special type of cancer a patient is suffering from, it is crucial to find the most efficient anti-cancer drug. The response of cancer cells to the different types of drugs is not entirely predictable though. Therefore a biopsy is taken, the cells are cultured and treated with different drugs. At the same time they are repeatedly imaged by FLIM. Fluorescence lifetimes indicate early shifts in the metabolic state of a cell after the treatment. Thus, with these measurements the most efficient medication can be determined within only a few days.

Combine Time-Domain Analysis with a Phasor Plot

The new generation of bh’s data analysis FLIM software combines time-domain multi-exponential decay analysis with a phasor plot. The measurement of fluorescence decay in the time-domain requires short excitation pulses and fast detection circuits. Each point in the sample is excited sequentially. With the time-domain methods, lifetimes are derived from exponential fits to the decay data.

The typical FLIM approach consists in extracting 1-, 2- or 3-decay times from the confocal image of the region of interest (ROI). Limiting the decay rates of fluorophores might be arbitrary and complicated, as there are several decay rates present in a cellular environment. With a phasor plot, the challenging decision on which decay model should be chosen and the evaluation of goodness of the fit will be a thing of the past. In a phaser plot all raw FLIM data is represented in a vector space as phase and amplitude. Each pixel in the image is transformed to a point in the phasor plot. Independently of their location in the image, pixels with similar decay signature form clusters in the phasor plot. It is possible to select different phasor clusters in this array and to back-annotate the corresponding pixels in the time-domain FLIM images.

Moreover, the decay functions of the pixels within the selected phasor range can be combined into a single decay curve of a high number of photons. This curve can be analysed at high accuracy, revealing decay components that are not visible by normal pixel-by-pixel analysis.

Bh’s SPCImage FLIM data analysis software uses a maximum-likelihood algorithm to calculate the parameters of decay functions in the individual pixel and accelerates by GPU processing the analysis procedure. 1D and 2D parameter histograms are available to display the distribution of all desired parameters over the pixels of the image or over selectable ROIs.

Leading Technology for All Your FLIM Needs

Choose the ideal equipment for your laboratory or clinic from our complete high-grade FLIM systems or our versatile upgrades for your existing microscope and scanner. You need further information on which solution would be best for your desired application? Send us an inquiry via our contact form or call us at +49 (30) 212 80 02-0!